Parkinson's disease (PD) is a common and fatal neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein a-synuclein, its impaired degradation, and oxidant damage are all hypotheses for the molecular cause of this selective neurotoxicity. In published findings with budding yeast (Sharma et al. 2006) and fission yeast (Brandis et al. 2006) models for a-synuclein misfolding and toxicity, we provided genetic and live cell support for these hypotheses, while uncovering unexpected yeast-specific a-synuclein property differences. In budding yeast, expression of a-synuclein alone does not cause toxicity, but the addition of either proteasomal dysfunction or mitochondrial oxidative stress is synthetic lethal. This lethality does not correlate well with a-synuclein aggregation. Instead, a-synuclein localizes primarily to the yeast plasma membrane even when toxic. In fission yeast, a-synuclein misfolds and aggregates within the cytoplasm in an exquisitely time and concentration-dependent manner, providing crucial live cell evidence for a mechanism that follows the nucleation-polymerization model. Despite the extensive aggregation, a-synuclein is not toxic. Even at low concentrations, it does not localize to the plasma membrane. By continuing such comparative analysis using both yeast models and by employing genetic manipulation in living cells, the following specific questions that all centrally focus on the molecular determinants within a-synuclein and cellular pathways that regulate its misfolding, lipid binding, degradation, and toxicity can be examined. What is the significance of the newly discovered familial PD mutation E46K in vivo? Does a-synuclein membrane localization in vivo involve specific phospholipids and is membrane interaction required for in vivo toxicity? Does a- synuclein contain domains that confer plasma membrane localization and aggregation in vivo? Can cytoplasmic oxidative stress also cause a-synuclein-mediated lethality or is lethality limited to mitochondrial stress? Lastly, does the lysosome also degrade a- synuclein and by what mechanism? The aims are: 1) To test the hypothesis that a-synuclein mutant E46K is significantly toxic to cells and binds phospholipids in vivo, E46K will compared to wild-type, A30P, A53T, and combination familial mutants (E46K/A53T, A30P/E46K, and A30P/E46K/A53T), in localization, aggregation, and toxicity in both yeasts. 2) To test the hypothesis that specific phospholipid composition and total phospholipid content is critical to a-synuclein membrane association and toxicity, a- synuclein localization (via GFP microscopy), aggregation (via GFP microcopy, Western blotting, and differential centrifugation), and toxicity (via growth curves, serial plating, and yeast cell death stains) will be evaluated in budding yeasts genetically compromised for the production of the major membrane phospholipids, and in both yeasts, supplemented with fatty acids or DMSO to increase overall phospholipid content. 3) To test the hypothesis that specific aggregation domains and lipid-binding domains mediate a-synuclein properties, N- and C-terminus fragments, along with specific point mutants will be tested for their localization, aggregation, and toxicity in both yeast models. 4) To test the hypothesis that cytoplasmic oxidative stress also contributes to a- synuclein-mediated toxicity, a-synuclein's localization, aggregation, and toxicity will be evaluated in budding yeasts compromised for major cytoplasmic antioxidant enzymes, including catalases, glutathione regulating enzymes, and DJ1, using single knockout strains, and multiple knockouts, where available. 5) To test the hypothesis that the lysosome pathway also degrades a-synuclein, the localization, aggregation, and toxicity of a-synuclein will be evaluated in budding yeast strains knocked out for genes encoding proteins that make up the well-studied multivesicular body (MVB) pathway to the yeast vacuole, which serves as its lysosome. Pilot undergraduate projects underway have already provided initial evidence to support some of these hypotheses. Once completed, these related studies, intentionally diverse in scope and designed to attract a diverse and large set of undergraduates to my lab, will together clarify the molecular bases for the regulation of the normal biology and pathobiology of a-synuclein. They will also expand the usefulness of multiple yeast models to study diverse protein misfolding diseases. Project Description Page 7 Principal Investigator/Program Director (Last, first, middle): DebBurman, Shubhik, Kumar PROJECT RELEVANCE The growing list of neurodegenerative diseases, including Parkinson's disease, represents a burgeoning public health concern in the United States. None of them are curable and most are fatal. Despite symptom diversity, many of these diseases share a common mechanism of pathology. Therefore, discoveries made with this Parkinson's disease grant may also impact progress for the other diseases. Undergraduate educators in the United States face significant challenges in preparing diverse graduates for a technologically sophisticated and scientifically interdisciplinary 21st-century community. Curricula that integrate more research and research-like experiences for non-majors and science majors in lieu of cookbook laboratory experiences, graduate a more diverse and larger group of well-prepared undergraduates that enter the scientific workforce. A major focus of this proposal is to provide rigorous scientific training to many undergraduates headed for future biomedical careers. Therefore, this proposal's major relevance is that it seeks to engage talented and diverse undergraduates in substantial and original research experiences, where they will contribute as scientists to biomedical discoveries linked to a major public health concern. 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